Introduction: The use of TLR9 agonists as immunomodulators is supported by preclinical and clinical studies, showing their anti-tumor effect by enhancing both cellular and humoral responses. Two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established so far: Dumbbell-shaped dSLIM molecules are protected against exonucleolytic degradation by their covalently-closed, natural phosphodiester (PO) backbone. In contrast, single-stranded, oligodeoxynucleotides (CpG-ODN) are most commonly chemically-stabilized by phosphorothioates (PTO) in their phosphate moieties. However, PTO modifications produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio.
Methods: Linear single-stranded ODN were synthesized using L-deoxyribonucleotides at their 3'-ends, which are the natural enantiomers of D-deoxyribonucleotides, to ensure protection against exonucleases and avoid the off-target effects of PTO-modified CpG-ODN. The vast majority of deoxyribose in present organisms are D-deoxyribose, thus co-evolved nucleases are blind for L-deoxyribose thereby leaving L-protected ODN intact. Using high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells as marker, we selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM. First, we employed a maximum feasible dose (MFD) approach. Mice received subcutaneous injection of single doses of 10 to 50 mg EnanDIM to evaluate its acute toxicity. Immunomodulatory properties were evaluated as well. Finally, a pilot tumor model in mice was used to investigate the anti-tumor effect of EnanDIM.
Results: EnanDIM581 and EnanDIM532 were selected due to their pronounced activation of immune cells (e.g. monocytes, NK cells and plasmacytoid dendritic cells) and their prominent induction of IFN-alpha and IP-10 secretion in vitro. EnanDIM744, comprising EnanDIM581 with additional 5'-end L-nucleotide protection and exhibiting an activation pattern similar to EnanDIM581, was also used for MFD studies. Safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the extremely high doses of app. 300 to 1700 mg/kg. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Dose-dependent increase of IP-10 levels in serum was observed between 6 and 24 hours after injection (maximum at 6h) but after 15 days IP-10 levels were undetectable, confirming that L-nucleotides in EnanDIM do not change the kinetic profile known from other DNA-based TLR9 agonists. Preliminary data from a murine tumor model suggest that multiple doses of EnanDIM can reduce tumor growth.
Conclusions: EnanDIM, a new family of TLR9 agonists, broadly activates the immune system in vitro and in vivo. While maximal feasible doses of EnanDIM resulted in no signs of toxicity, an indication of reduced tumor growth was observed in a murine tumor model in vivo. Therefore EnanDIM has the potential for clinical development in the treatment of cancer.
Citation Format: Kerstin Kapp, Barbara Volz, Detlef Oswald, Burghardt Wittig, Manuel Schmidt. Preclinical data of novel enantiomeric oligonucleotides for cancer immunotherapy: The TLR9 agonist EnanDIM [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B067.
- ©2016 American Association for Cancer Research.